Project Summary The goal of this project is to determine structural mechanisms by which for the parathyroid hormone (PTH) receptor (PTHR) signals in response to its functionally distinct ligands: PTH, PTH-related peptide (PTHrP) and the long-acting PTH analog (LA-PTH). The PTHR is a major G protein-coupled receptor (GPCR) that regulates Ca2+ homeostasis in blood and bone turnover, and is the most effective therapeutic target for osteoporosis. It also is one of the first GPCR found to sustain cAMP production after internalization of the PTH?receptor complex in endosomes. The recently recognized feature that the calcemic action of PTHR in mice and primates is sustained by LA-PTH, which also prolongs endosomal cAMP production, is changing our thinking about how PTHR mediates its physiological actions. Implicit in these findings is that efficient treatment of hypocalcemia might be more approachable with selective targeting of PTHR-mediated endosomal cAMP signaling. The mechanism that differentiating the signaling selectivity of PTH and its analogs are not known and this is an obstacle to further move toward new directions to develop PTH-based therapies that have improved efficacies for treating bone and mineral diseases. We therefore propose a research program to overcome this obstacle. The goal of this project is thus to determine the structural basis by which PTHR function and activate G proteins in response to PTH, PTHrP and LA-PTH. Two specific aims are proposed to discover structural basis of PTHR signaling. The first aim addresses the hypothesis that PTH and LA-PTH promote long endosomal cAMP production by stabilizing unique structural arrangements and phosphorylation pattern in the PTHR that are different from those stabilized by PTHrP, which induces a short cAMP response from the plasma membrane. To this end, we will use quantitative mass spectrometry (MS) based-proteomics technologies, such as Hydrogen-Deuterium exchange coupled to MS (HDXMS) to determine structural changes and structural determinants for PTHR activation upon binding to the distinct ligands, and identify the binding interface in PTHR?G protein complexes. The second aim will complement our understanding of the functional selectivity of PTHR by X-ray crystallography of PTHR and PTHR bound to PTH, PTHrP and LA-PTH. We have obtained X-ray diffractable crystals (4 ) of the full length PTHR bound to LA-PTH and in complex with the RNA polymerase II. Here we are using the cavity formed in the RNA polymerase II crystal as a ?crystal sponge? that creates a favorable crystallization environment for PTHR. We will initially optimize the crystallization conditions to resolve crystal structure of the PTHR bound to LA-PTH in complex with RNA polymerase II at higher resolution (2-3 ). We will then focus of resolving PTH and PTHrP-bound states of PTHR. These studies will provide new insights into how PTHR activate G proteins and the structural mechanism differentiating the action of PTH, PTHrP and LA-PTH.